LC-MS/MS分析人参茎叶提取液中人参皂苷Rg1、Re降解产物

目的研究人参茎叶提取液中人参皂苷Rg1、Re加热后所产生的降解产物,并分析产物的结构。方法采用液相色谱/离子阱质谱联用仪对降解产物进行分析,并推测其结构,HPLC的条件为Agilent Extend-C18(250 mm×4.6 mm,5μm)色谱柱,流动相为乙腈-水,梯度洗脱70 min,检测波长203 nm,流速0.4 mL·min^-1,柱温30℃;质谱条件为正离子检测方式,毛细管电压3 000 V,喷雾器15 psi,干燥器流速12.0 L·min^-1,干燥器温度350℃,扫描范围100~1 000 m/z;碎裂器范围4 500~1 500 V。结果质谱图中有多种化合物,推测人参皂苷Rg1、Re在人参茎叶提取液的pH值环境下进行加热,会发生脱糖、脱羟基、环合等反应,生成多种降解产物。结论人参皂苷水溶液在人参茎叶提取液pH环境下加热的时间与温度对人参皂苷的降解非常敏感,说明此方法适用于人参皂苷类化合物的降解与分析。     

Simultaneously determination of five ginsenosides in rabbit plasma using solid-phase extraction and HPLC/MS technique after intravenous administration of 'SHENMAI' injection

In this study, a sensitive and reliable analytical method for the simultaneous determination of five ginsenosides (R(g1), R(f), R(e), R(d) and R(b1)) in rabbit plasma was developed by high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS). Chromatographic separation was carried out on a Zorbax SB-C18 Column (150 mm x 2.1 mm i.d., 5.0 microm particle size) with a simple linear gradient elution. The detection was conducted on a single quadrupole mass spectrometer by selected ion monitoring mode via electrospray ionization source. Good linearity over the investigated concentration ranges was observed with the values of R(2) higher than 0.991 for all the analytes. Limits of detection of the analytes varied from 0.25 ng/ml to 1.45 ng/ml, and the average recoveries, examined at three concentration levels, ranged from 90.6% to 106.9%. The validated method was successfully applied to the determination of the ginsenosides in the rabbit plasma after intravenous administration of 'SHENMAI' injection.     

基于UHPLC-HRMS/MS的鲜人参不同部位中皂苷类成分差异分析

目的 采用UHPLC-HRMS/MS对鲜人参中的三萜皂苷类成分进行分析鉴定,并阐明鲜人参不同部位(主根、侧根、须根和芦头)所含的三萜皂苷类成分的差异性。方法 以水饱和正丁醇为提取溶剂,以水-乙腈为流动相进行梯度洗脱,在ESI负离子模式下建立新鲜人参药材中皂苷类成分的UHPLC-Q-Exactive Orbitrap MS/MS分析表征方法,并采集各样品的高分辨质谱数据。结合色谱保留行为、精确分子量、多级质谱裂解规律及对照品比对等,鉴定各样品中的人参皂苷类成分;依据已鉴定的人参皂苷色谱峰的相对丰度,筛选差异色谱峰,阐明鲜人参各部位差异。结果 从鲜人参不同部位中共检测到68种差异皂苷类成分,其中主根有44种,侧根有47种,须根有52种,芦头有37种。结论 鲜人参不同部位中皂苷类成分差异较大;人参侧根、芦头等非主要药用部位中含有多种人参皂苷,且存在其他部位未检测到的成分,可作为药物研发的重要原料及多种人参皂苷的提取原料。     

红参中总皂苷及人参皂苷Rg1、Re、Rb1

目的测定不同产地的26批红参药材中总皂苷及人参皂苷Rg₁、Re、Rb₁的含量。方法采用紫外可见分光光度法测定红参中总皂苷的含量,高效液相色谱法测定红参中人参皂苷Rg 1、Re、Rb 1的含量。结果26批红参药材中总皂苷含量为1.2%~3.0%,人参皂苷Rg₁、Re含量之和为0.17%~0.61%,人参皂苷Rb₁含量为0.21%~0.81%。结论不同产地的红参药材总皂苷及人参皂苷Rg₁、Re、Rb₁的含量存在差异,提示应进一步规范红参药材的种植、加工炮制及标准的建立。     

LC/MS fingerprinting of Shenmai injection: a novel approach to quality control of herbal medicines

Chromatographic fingerprinting has been recommended as a potential and reliable strategy for the quality control of herbal medicines. Although varieties of chromatographic techniques, particularly HPLC, have been widely employed, hyphenated chromatographic approach has not been sufficiently exploited in chromatographic fingerprinting. In this work, LC/MS fingerprinting of Shenmai injection was developed. Thirty ginsenosides as well as seven ophioponins were selected to construct the LC/MS fingerprint using selective ion monitoring (SIM) mode, while previous HPLC fingerprint [H.J. Zhang, Y.J. Wu, Y.Y. Cheng, J. Pharm. Biomed. Anal. 31 (2003) 175-183] only represents the ginsenosides. Subsequently, the proposed LC/MS fingerprints were applied to identifying the product manufacturers. All the samples were accurately classified based on their LC/MS fingerprints in conjunction with principal components analysis (PCA). This study would be potentially helpful to improve the quality control ability of fingerprinting-based strategy for complex herbal medicines.     

Decocting-induced chemical transformations and global quality of Du–Shen–Tang,the decoction of ginseng evaluated by UPLC–Q-TOF-MS/MS based chemical profiling approach

An UPLC–Q-TOF-MS/MS based chemical profiling method was developed to evaluate decocting-induced chemical transformations in Du–Shen–Tang, the decoction of the root of Panax ginseng. Under the optimized UPLC and Q-TOF-MS/MS conditions, over 50 peaks were separated and detected in Du–Shen–Tang within 18 min. The components were identified by comparing the mass spectra and retention time with that of reference compounds, and/or tentatively assigned by elucidating low energy CID fragment ions as well as matching empirical molecular formula with that of the published known compounds. Totally 45 major ginsenosides were identified in Du–Shen–Tang, 21 of which were determined to be newly generated during the decoction of ginseng. The mechanisms involved were further deduced to be hydrolysis, dehydration, decarboxylation and addition reactions of the original ginsenosides in white ginseng through analyzing mimic decoctions of 13 pure reference ginsenosides. Significant difference in chemical profiles between decoctions of two batches of white ginseng suggested that storage duration or other factors significantly influenced the quality consistency of not only the crude drug but also the decoction (Du–Shen–Tang) of white ginseng.     

Comparative study on intestinal metabolism and absorption in vivo of ginsenosides in sulphur‐fumigated and non‐fumigated ginseng by ultra performance liquid chromatography quadruple time‐of‐flight mass spectrometry based chemical profiling approach

Our previous study indicated that sulphur‐fumigation of ginseng in post‐harvest handling processes could induce chemical transformation of ginsenosides to generate multiple ginsenoside sulphur derivatives. In this study, the influence of sulphur‐fumigation on intestinal metabolism and absorption in vivo of ginsenosides in ginseng was sequentially studied. The intestinal metabolic and absorbed profiles of ginsenosides in rats after intra‐gastric (i.g.) administration of sulphur‐fumigated ginseng (SFG) and non‐fumigated ginseng (NFG) were comparatively characterized by a newly established ultra performance liquid chromatography quadruple time‐of‐flight mass spectrometry (UPLC‐QTOF‐MS/MS) with electrospray ionization negative (ESI‐) mode. A novel strategy based on the characteristic product ions and fragmentation pathways of different types of aglycones (saponin skeletons) and glycosyl moieties was proposed and successfully applied to rapid structural identification of ginsenoside sulphur derivatives and relevant metabolites. In total, 18 ginsenoside sulphur derivatives and 26 ginsenoside sulphur derivative metabolites in the faeces together with six ginsenoside sulphur derivatives in the plasma were identified in the SFG‐administrated group but not in the NFG‐administrated group. The results clearly demonstrated that the intestinal metabolic and absorbed profiles of ginsenosides in sulphur‐fumigated and non‐fumigated ginseng were quite different, which inspired that sulphur‐fumigation of ginseng should not be recommended before the bioactivity and toxicity of the ginsenoside sulphur derivatives were systematically evaluated. Copyright © 2014 John Wiley & Sons, Ltd.     

Polyacetylenes in hairy roots of a panax hybrid

From the hairy roots of an interspecific hybrid ginseng ( Panax ginseng x P. quinquefolium) known as Pgq, three polyacetylenes were isolated: panaxynol, panaxydol and 1,8-heptadecadiene-3,10-diol. These compounds isolated from the hairy roots were used for quantitative analysis to investigate the polyacetylene production of the hairy roots cultured in Gamborg B5 (B5) and 1/8 Murashige-Skoog (MS) liquid media. Maximum growth of the hairy roots was observed (ca. 5.4 g fresh weight/100 mL flask) at 8 weeks of culture in B5 medium. The highest total content of total polyacetylenes was 0.18 % of dry weight at week 8 when cultured in 1/8 MS medium. In addition, we compared the yields of polyacetylenes and ginsenosides in hairy roots cultured in B5 with those in 1/8 MS media and found the highest yields were obtained in the hairy roots cultured in B5 medium (1.24 mmol/flask polyacetylenes and 4.45 mmol/flask ginsenosides at week 8).     

LC/MS鉴定中药三七及其复方制剂

目的建立中药三七及其在复方丹参制剂中的特征图谱。方法用LC/MS分析方法,采集三七、人参和西洋参的LC/MS总离子流色谱,从总离子流色谱中提取三七主要化学组分的提取离子流色谱,选择相对稳定、3种药材间差异显著的提取离子流色谱作为三七药材的特征图谱。在相同条件下对比复方丹参的提取离子流色谱与三七的特征图谱;同时用LC/MS/MS对三七中的主要组分进行定性分析。结果与人参皂苷Rg1(m/z 800)和Re(m/z 946)具有相同m/z的提取离子流色谱在3种药材间差异显著,稳定可靠,在复方中有较好的重现性,复方丹参中其他共有成分对其无显著影响。结论三七的LC/MS特征图谱可作为鉴定复方丹参中三七的特征,也可作为鉴别三七和同属其他药材的特征。     

HPLC-MS/MS法同时测定三七须根总皂苷中人参皂苷Rg_1和Re的含量

目的:建立以液-质联用法同时测定三七须根总皂苷中人参皂苷Rg1和Re含量的方法。方法:色谱柱为BDS HYPERSUL C18(150mm×2.1mm,5μm),流动相为乙腈-水(梯度洗脱),柱温为30℃;采用负离子多反应监测方法(MRM)测试。用于定量分析的对照品离子对分别为人参皂苷Rg1:m/z799.6→475.5;人参皂苷Re:m/z945.8→783.7;内标物紫杉醇m/z:852.5→525.3。结果:人参皂苷Rg1、人参皂苷Re标准曲线的线性范围分别为0.173～17.3μg·mL-1和0.156～39.1μg·mL-1,精密度和准确度等均符合样品分析的要求。结论:本方法准确、灵敏、特异性强,适用于三七须根总皂苷及其制剂中人参皂苷Rg1、Re含量的同时测定。     

Chemical investigation on Sijunzi decoction and its two major herbs Panax ginseng and Glycyrrhiza uralensis by LC/MS/MS

Sijunzi decoction consists of Panax ginseng, Poria cocos, Atractylodes macrocephala and Glycyrrhiza uralensis. High performance liquid chromatography coupled with tandem mass spectrometry (LC/MSⁿ) was applied to identify and characterize three types of active components, ginsenoside (from P. ginseng), flavonoid and triterpenoid (from G. uralensis) in Sijunzi decoction. Spectra of MS and MS/MS from [M + Na]⁺ ions of ginsenosides were acquired and interpreted for their identification. Fragmentations with losing masses of 194 or 176 Da were the characteristic ions of triterpenoids in the MS/MS analysis. A characteristic fragment ion of the aglycon moiety at m/z 257 from source collision-induced dissociation was observed for flavonoid. LC/MS was also applied for the comparison of relative peak area of major active components between Sijunzi decoction and the single herb extracts. The concentration ratios of major active components detected in the individual herbs of P. ginseng and G. uralensis were found different from those in Sijunzi decoction. The experimental data indicated that the decocting process could result in the difference in the amount of active components.     

Stimulatory effect of ginsenosides on DNA, protein and lipid synthesis in rat bone marrow and participation of cyclic nucleotides.

Effects of several kinds of ginsenosides, saponins from Panax ginseng on DNA, RNA, protein and lipid synthesis in bone marrow were investigated. Single i.p. injection of 0.5--1 mg/100 g body weight of ginsenosides Rb2, Rc, Re and Rg1 4 h prior to the sacrifice increased DNA synthesis in bone marrow cells. RNA, protein and lipid synthesis were also increased. The direct addition of ginsenosides Rb1, Rb2 and Rc mixture (GNS) enhanced DNA synthesis. Cyclic AMP levels in bone marrow cells were decreased 20 min after i.p. injection of ginsenosides Rb2, Rc and Rg1 and the direct addition of ginsenosides Rb2, Rc and Rg1 also decreased cyclic AMP levels. While cyclic GMP levels were increased by administration of ginsenosides Rb2, Re and Rg1. Relationship between chemical structure and actions of ginsenosides and the role of cyclic nucleotides in the stimulatory action of ginsenosides on DNA synthesis in bone marrow cells were discussed.     

The inhibitory effects of ginsenosides on protein tyrosine kinase activated by hypoxia/reoxygenation in cultured human umbilical vein endothelial cells

27 individual ginsenosides and aglycones, together with five extracts from ginseng roots, ginseng leaves, American ginseng roots, American ginseng leaves and non-saponin fraction from roots of Panax ginseng, were tested for their effects on protein tyrosine kinase (PTK) activation induced by an in vitro hypoxia/reoxygenation (H/R) model in cultured human umbilical vein endothelial cells (HUVEC). The results indicated that ginsenoside-Rb1 (3), -Rd (7), -Ra1 (1) and -Ro (27) showed significant inhibitory effects on PTK activation induced by H/R. Dose-response experiments revealed that ginsenoside-Rb1 was the most active compound and it completely blocked PTK activation at a wide range of concentrations. Most protopanaxadiol-type ginsenosides and some protopanaxatriol-type saponins also showed significant effects on PTK activation. However, the crude extracts did not protect against H/R-induced PTK activation.     

扣子七中人参皂苷的HPLC-MS-MS方法研究

扣子七为五加科人参届植物大叶三七Panaxjaponicus C.A.Mey.var.major(Burkill)C.Y.Wu et K.M.Feng的干燥根茎,分布于云南,重庆,湖北等地。在渝东鄂西,扣子七用于养阴,清肺,散淤,止血,定痛,其化学成分的研究尚未见报道。 本实验以HPLC分离扣子七中人参皂苷,用离     

In vivo antimalarial activity of ginseng extracts

Context: Novel antimalarial agents are in demand due to the emergence of multidrug resistant strains. Ginseng, a medicinal plant with antiparasitic activity, contains components that can be used to treat the tropical disease malaria.

Objective: Ginsenosides and polysaccharides are active components of ginseng. This study aimed to elucidate the ability of these compounds to inhibit the replication of Plasmodium yoelii in an attempt to determine whether the medicinal uses of ginseng are supported by pharmacological effects. New antimalarial compounds may be developed from ginsenosides and water-soluble ginseng polysaccharides (WGP).

Materials and methods: Ginsenosides and ginseng polysaccharides were prepared from ginseng. Antimalarial activities were examined by 4-day tests and repository tests. Macrophage phagocytosis was tested in normal and malaria-bearing mice.

Results: Ginseng polysaccharides could inhibit residual malaria infection. After a 6-day treatment, the parasitemia reductions of WGP and acidic ginseng polysaccharide (WGPA) were 55.66% and 64.73% at 200?mg/kg/day, respectively. Ginsenosides showed significant antimalarial activity on early infection. Protopanaxadiol-type ginsenosides caused 70.97% chemosuppression at 50?mg/kg/day, higher than 52.8% of total ginsenosides at the same dose.

Discussion and conclusion: Protopanaxadiol-type ginsenosides have remarkably suppressive activity during early infection, while acidic ginseng polysaccharides have significant prophylactic activity against malaria by stimulating the immune system. We propose that the activity of ginsenosides is dependent upon non-specific carbohydrate interactions and that the activity of ginseng polysaccharides is due to immunological modulation. Ginsenosides and ginseng polysaccharides might have a potential application in antimalarial treatments.     

Biotransformation of Korean Panax ginseng by Pectinex

Ginsenosides comprise the major component of ginseng exhibit various types of biological activity, including antiinflammatory and antitumor effects. In these pharmacological actions, it is thought that these activities are carried out by the metabolites of ginsenosides metabolized by human intestinal microflora. It has also been reported that their clinical efficacy varies with the hydrolyzing potential of the components of the intestinal microflora. We tried to develop a process for metabolizing ginsenosides to compound K using food-grade enzymes, which can be used commercially. Among these, Pectinex proved to be the most effective mediator of the catabolism of ginsenosides to compound K. The optimal conditions for this biotransformation were determined to be as follows: 10 to 15% rootlet ginseng, pH 5, 50 degrees C, and 2 to 3 d of incubation, to yield 20.0 mg of compound K/g of rootlet ginseng. We suggest that the metabolism of ginseng to compound K in the presence of Pectinex has many advantages over previous methods, in respects of use of raw, non-extracted rootlet ginseng, which do not require more organic solvents and evaporation apparatus. Potential metabolites PG1, PG2, PG3, and PG4 were detected in Pectinex-treated rootlet ginseng using by TLC and HPLC and, among them, PG4 was identified as compound K by TLC, HPLC, and MS. Additional studies will be carried out to determine the structure of these metabolites of ginseng and to understand the relationship between their structures and activities.     

Chemical investigation on Sijunzi decoction and its two major herbs Panax ginseng and Glycyrrhiza uralensis by LC/MS/MS

Sijunzi decoction consists of Panax ginseng, Poria cocos, Atractylodes macrocephala and Glycyrrhiza uralensis. High performance liquid chromatography coupled with tandem mass spectrometry (LC/MSⁿ) was applied to identify and characterize three types of active components, ginsenoside (from P. ginseng), flavonoid and triterpenoid (from G. uralensis) in Sijunzi decoction. Spectra of MS and MS/MS from [M + Na]⁺ ions of ginsenosides were acquired and interpreted for their identification. Fragmentations with losing masses of 194 or 176 Da were the characteristic ions of triterpenoids in the MS/MS analysis. A characteristic fragment ion of the aglycon moiety at m/z 257 from source collision-induced dissociation was observed for flavonoid. LC/MS was also applied for the comparison of relative peak area of major active components between Sijunzi decoction and the single herb extracts. The concentration ratios of major active components detected in the individual herbs of P. ginseng and G. uralensis were found different from those in Sijunzi decoction. The experimental data indicated that the decocting process could result in the difference in the amount of active components.     

枇杷花的研究进展及其药效评价思路的探讨

枇杷花具有重要的药用价值和开发潜力.近年来国内外相关研究主要集中在枇杷花的化学成分、提取和精制工艺、药理效应和应用开发等方面,但存在有效性评价方法不系统和有效剂量不明确等问题.在综述枇杷花化学成分、提取和精制工艺、药理作用的基础上,提出如下研究思路:基于传统用药经验,应用经典、快速的动物模型确定有效剂量范围,整合多致病...     

基于UPLC-TOF/MS分析人参附子配伍减毒的物质基础

利用超高效液相色谱-飞行时间质谱联用技术(UPLC-TOF/MS)分析人参附子药对配伍减毒的物质基础,从化学成分层次阐释其配伍减毒机制。基于UPLC-TOF/MS建立人参附子药对配伍后生物碱类成分的化学指纹图谱,通过主成分分析法和正交偏最小二乘判别法分析药对配伍在合煎过程中的生物碱类成分的含量变化,找出差异变化显著的化学成分。结果表明,正离子模式时人参附子药对合煎液中次乌头碱、去氧乌头碱的含量明显降低,而苯甲酰中乌头原碱、苯甲酰次乌头原碱和去乙酸中乌头原碱等含量升高。人参附子药对配伍应用时双酯型二萜生物碱的含量明显降低,而单酯型二萜生物碱的含量明显升高,这可能是人参附子药对配伍减毒作用的物质基础。     

The combination effect of Korean red ginseng saponins with kanamycin and cefotaxime against methicillin-resistant Staphylococcus aureus

Korean red ginseng saponins (ginsenosides) have been reported as having various biological properties, but the combinational effects with commercial antibiotics and the mode of action of ginsenosides remain mostly unknown. In this study, saponins were isolated from Korean red ginseng, and the antibacterial effects of ginsenosides were investigated. Ginsenosides showed antibacterial activities toward pathogenic Gram-positive and Gram-negative bacteria. To elucidate the antibacterial mode of action of ginsenosides, we measured the release of the fluorescent marker calcein from negatively charged PC/PG (1 : 1, w/w) liposomes, which mimic bacterial membranes. The results suggest that ginsenosides may exert antibacterial activity by disrupting the cell membrane. To estimate the general combination effects of ginsenosides and commercial antibiotics, such as kanamycin and cefotaxime, on antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) strains that were clinically isolated from an infected patient, the fraction inhibitory concentration (FIC) indexes were determined by a checkerboard study. The FIC indexes showed synergistic or additive effects between the ginsenosides and antibiotics tested.